The following description of the background of the invention is provided simply as an aid in understanding the invention and is not admitted to describe or constitute prior art to the invention.
As discussed in Fredline et al. Clin. Chem. 45: 659-664 (1999), renin is a proteolytic enzyme secreted into blood by the juxtaglomerular cells of the kidney. Renin acts on angiotensinogen, to produce a decapeptide referred to as angiotensin 1 (Ang1). Ang1 is further cleaved by angiotensin-converting enzyme to form an octapeptide, referred to as angiotensin 2 (Ang2). Ang2 stimulates cell growth, renal tubule transport of sodium, and aldosterone release. Ang2 is one of the most potent vasopressors in humans and plays an important role in blood pressure regulation. Direct measurement of Ang2 is difficult because of its very low circulating concentrations and extremely short half-life: Ang1, which is more stable than Ang2, provides a better analyte to measure the state of the renin-angiotensin system. Determination of a “plasma renin activity” (PRA) from the rate of generation of Ang1 is used clinically for the diagnosis and management of hypertension.
The classical method for the determining PRA is radioimmunoassay (RIA), see Sealey, Clin. Chem. 37:1811-1819 (1991); Shionoiri et al., Horm Res. 37:171-175 (1992). A typical radioimmunoassay is performed by the simultaneous preparation of a series of standard and unknown mixtures in test tubes, each containing identical concentrations of labeled antigen and specific antibody. After an appropriate reaction time, the antibody-bound (B) and free (F) fractions of the labeled antigen are separated by one of a variety of techniques. The B/F ratios in the standards are plotted as a function of the concentration of unlabeled antigen (standard curve), and the unknown concentration of antigen is determined by comparing the observed B/F ratio with the standard curve. Radioimmunoassay methods are based on competitive binding principles, and the antibodies used can undergo nonspecific binding with other plasma proteins such as endogenous angiotensins. This potential cross-reactivity can cause overestimation of the PRA. Another approach is to use HPLC to isolate Ang1 from other angiotensins before quantification with RIA (see examples from Meng et al., J. Am. Soc. Nephrol. 6: 1209-1215 (1995); Meng et al., J. Chromatogr. 21, 614(1): 19-25 (1993); Kohara et al., Peptides 12: 1135-1141 (1991)). These HPLC methods for the quantification of Ang1 have been developed using ultraviolet, fluorescence and mass spectrometer detection (see examples from Klickstein et al. Anal Biochem 120: 146-150 (1982); Miyazaki et al. J. Chromatogr. 490: 43-51 (1989) and Fredline et al. Clin. Chem. 45: 659-664 (1999)).
For example, Fredline et al. describes measurement of plasma renin activity with the use of HPLC-electrospray-tandem mass spectrometry. In doing this measurement, Fredline et al. incubates plasma samples in the presence of a water-sensitive enzyme inhibitor (i.e., phenylmethylsulphonyl fluoride (PMSF)) and measures the amount of angiotensin 1 generated during incubation by observing the collision induced dissociation of precursor ions with a (m/z) of 649.